Optical Control of G-Actin with a Photoswitchable Latrunculin.

Actin is one of the most abundant proteins in eukaryotic cells and is a key component of the cytoskeleton. A range of small molecules has emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Among these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390-490 nm pulsed light and rapidly relaxes to its inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated via live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of the microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

Chaperone-mediated autophagy in neuronal dendrites utilizes activity-dependent lysosomal exocytosis for protein disposal.

The complex morphology of neurons poses a challenge for proteostasis because the majority of lysosomal degradation machinery is present in the cell soma. In recent years, however, mature lysosomes were identified in dendrites, and a fraction of those appear to fuse with the plasma membrane and release their content to the extracellular space. Here, we report that dendritic lysosomes are heterogeneous in their composition and that only those containing lysosome-associated membrane protein (LAMP) 2A and 2B fuse with the membrane and exhibit activity-dependent motility. Exocytotic lysosomes dock in close proximity to GluN2B-containing N-methyl-D-aspartate-receptors (NMDAR) via an association of LAMP2B to the membrane-associated guanylate kinase family member SAP102/Dlg3. NMDAR-activation decreases lysosome motility and promotes membrane fusion. We find that chaperone-mediated autophagy is a supplier of content that is released to the extracellular space via lysosome exocytosis. This mechanism enables local disposal of aggregation-prone proteins like TDP-43 and huntingtin.

Optical Control of G-Actin with a Photoswitchable Latrunculin.

Actin is one of the most abundant proteins in eukaryotic cells and a key component of the cytoskeleton. A range of small molecules have emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Amongst these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390 - 490 nm pulsed light and rapidly relaxes to the inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated by live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

Assessment of Murine Retinal Acuity Ex Vivo Using Multielectrode Array Recordings.

Purpose: Visual acuity, measured by resolution of optotypes on a standard eye chart, is a critical clinical test for function of the visual system in humans. Behavioral tests in animals can be used to estimate visual acuity. However, such tests may be limited in the study of mutants or after synthetic vision restoration techniques. Because the total response of the retina to a visual scene is encoded in spiking patterns of retinal ganglion cells, it should be possible to estimate visual acuity in vitro from the retina by analyzing retinal ganglion cell output in response to test stimuli. Methods: We created a method, EyeCandy, that combines a visual stimulus-generating engine with analysis of multielectrode array retinal recordings via a machine learning approach to measure murine retinal acuity in vitro. Visual stimuli included static checkerboards, drifting gratings, and letter optotypes. Results: In retinas from wild-type C57Bl/6 mice, retinal acuity measurement for a drifting grating was 0.4 cycles per degree. In contrast, retinas from adult rd1 mice with outer retinal degeneration showed no detectable acuity. A comparison of acuities among different regions of the retina revealed substantial variation, with the inferior-nasal quadrant having highest RA. Letter classification accuracy of a projected Early Treatment Diabetic Retinopathy eye chart reached 99% accuracy for logMAR 3.0 letters. EyeCandy measured a restored RA of 0.05 and 0.08 cycles per degree for static and dynamic stimuli respectively from the retina of the rd1 mouse treated with the azobenzene photoswitch BENAQ. Conclusions: Machine learning may be used to estimate retinal acuity. Translational Relevance: The use of ex vivo retinal acuity measurement may allow determination of effects of mutations, drugs, injury, or other manipulations on retinal visual function.

ΔFosB accumulation in hippocampal granule cells drives cFos pattern separation during spatial learning.

Mice display signs of fear when neurons that express cFos during fear conditioning are artificially reactivated. This finding gave rise to the notion that cFos marks neurons that encode specific memories. Here we show that cFos expression patterns in the mouse dentate gyrus (DG) change dramatically from day to day in a water maze spatial learning paradigm, regardless of training level. Optogenetic inhibition of neurons that expressed cFos on the first training day affected performance days later, suggesting that these neurons continue to be important for spatial memory recall. The mechanism preventing repeated cFos expression in DG granule cells involves accumulation of ΔFosB, a long-lived splice variant of FosB. CA1 neurons, in contrast, repeatedly expressed cFos. Thus, cFos-expressing granule cells may encode new features being added to the internal representation during the last training session. This form of timestamping is thought to be required for the formation of episodic memories.

The role of microglia membrane potential in chemotaxis.

Microglia react to danger signals by rapid and targeted extension of cellular processes towards the source of the signal. This positive chemotactic response is accompanied by a hyperpolarization of the microglia membrane. Here, we show that optogenetic depolarization of microglia has little effect on baseline motility, but significantly slows down the chemotactic response. Reducing the extracellular Ca2+ concentration mimics the effect of optogenetic depolarization. As the membrane potential sets the driving force for Ca2+ entry, hyperpolarization is an integral part of rapid stimulus-response coupling in microglia. Compared to typical excitable cells such as neurons, the sign of the activating response is inverted in microglia, leading to inhibition by depolarizing channelrhodopsins.

Photopharmacologic Vision Restoration Reduces Pathological Rhythmic Field Potentials in Blind Mouse Retina.

Photopharmacology has yielded compounds that have potential to restore impaired visual responses resulting from outer retinal degeneration diseases such as retinitis pigmentosa. Here we evaluate two photoswitchable azobenzene ion channel blockers, DAQ and DAA for vision restoration. DAQ exerts its effect primarily on RGCs, whereas DAA induces light-dependent spiking primarily through amacrine cell activation. Degeneration-induced local field potentials remain a major challenge common to all vision restoration approaches. These 5-10 Hz rhythmic potentials increase the background firing rate of retinal ganglion cells (RGCs) and overlay the stimulated response, thereby reducing signal-to-noise ratio. Along with the bipolar cell-selective photoswitch DAD and second-generation RGC-targeting photoswitch PhENAQ, we investigated the effects of DAA and DAQ on rhythmic local field potentials (LFPs) occurring in the degenerating retina. We found that photoswitches targeting neurons upstream of RGCs, DAA (amacrine cells) and DAD (bipolar cells) suppress the frequency of LFPs, while DAQ and PhENAQ (RGCs) had negligible effects on frequency or spectral power of LFPs. Taken together, these results demonstrate remarkable diversity of cell-type specificity of photoswitchable channel blockers in the retina and suggest that specific compounds may counter rhythmic LFPs to produce superior signal-to-noise characteristics in vision restoration.

Sign Inversion in Photopharmacology: Incorporation of Cyclic Azobenzenes in Photoswitchable Potassium Channel Blockers and Openers.

Photopharmacology relies on ligands that change their pharmacodynamics upon photoisomerization. Many of these ligands are azobenzenes that are thermodynamically more stable in their elongated trans-configuration. Often, they are biologically active in this form and lose activity upon irradiation and photoisomerization to their cis-isomer. Recently, cyclic azobenzenes, so-called diazocines, have emerged, which are thermodynamically more stable in their bent cis-form. Incorporation of these switches into a variety of photopharmaceuticals could convert dark-active ligands into dark-inactive ligands, which is preferred in most biological applications. This "pharmacological sign-inversion" is demonstrated for a photochromic blocker of voltage-gated potassium channels, termed CAL, and a photochromic opener of G protein-coupled inwardly rectifying potassium (GIRK) channels, termed CLOGO.

Photopharmacological control of bipolar cells restores visual function in blind mice.

Photopharmacological control of neuronal activity using synthetic photochromic ligands, or photoswitches, is a promising approach for restoring visual function in patients suffering from degenerative retinal diseases. Azobenzene photoswitches, such as AAQ and DENAQ, have been shown to restore the responses of retinal ganglion cells to light in mouse models of retinal degeneration but do not recapitulate native retinal signal processing. Here, we describe diethylamino-azo-diethylamino (DAD), a third-generation photoswitch that is capable of restoring retinal ganglion cell light responses to blue or white light. In acute brain slices of murine layer 2/3 cortical neurons, we determined that the photoswitch quickly relaxes to its inactive form in the dark. DAD is not permanently charged, and the uncharged form enables the photoswitch to rapidly and effectively cross biological barriers and thereby access and photosensitize retinal neurons. Intravitreal injection of DAD restored retinal light responses and light-driven behavior to blind mice. Unlike DENAQ, DAD acts upstream of retinal ganglion cells, primarily conferring light sensitivity to bipolar cells. Moreover, DAD was capable of generating ON and OFF visual responses in the blind retina by utilizing intrinsic retinal circuitry, which may be advantageous for restoring visual function.

Optical control of GIRK channels using visible light.

G-protein coupled inwardly rectifying potassium (GIRK) channels are an integral part of inhibitory signal transduction pathways, reducing the activity of excitable cells via hyperpolarization. They play crucial roles in processes such as cardiac output, cognition and the coordination of movement. Therefore, the precision control of GIRK channels is of critical importance. Here, we describe the development of the azobenzene containing molecule VLOGO (Visible Light Operated GIRK channel Opener), which activates GIRK channels in the dark and is promptly deactivated when illuminated with green light. VLOGO is a valuable addition to the existing tools for the optical control of GIRK channels as it circumvents the need to use potentially harmful UV irradiation. We therefore believe that VLOGO will be a useful research tool for studying GIRK channels in biological systems.

Optical control of NMDA receptors with a diffusible photoswitch.

N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity, learning and memory, and are implicated in various neuronal disorders. We synthesized a diffusible photochromic glutamate analogue, azobenzene-triazole-glutamate (ATG), which is specific for NMDARs and functions as a photoswitchable agonist. ATG is inactive in its dark-adapted trans-isoform, but can be converted into its active cis-isoform using one-photon (near UV) or two-photon (740 nm) excitation. Irradiation with violet light photo-inactivates ATG within milliseconds, allowing agonist removal on the timescale of NMDAR deactivation. ATG is compatible with Ca(2+) imaging and can be used to optically mimic synaptic coincidence detection protocols. Thus, ATG can be used like traditional caged glutamate compounds, but with the added advantages of NMDAR specificity, low antagonism of GABAR-mediated currents, and precise temporal control of agonist delivery.

AzoCholine Enables Optical Control of Alpha 7 Nicotinic Acetylcholine Receptors in Neural Networks.

Nicotinic acetylcholine receptors (nAChRs) are essential for cellular communication in higher organisms. Even though a vast pharmacological toolset to study cholinergic systems has been developed, control of endogenous neuronal nAChRs with high spatiotemporal precision has been lacking. To address this issue, we have generated photoswitchable nAChR agonists and re-evaluated the known photochromic ligand, BisQ. Using electrophysiology, we found that one of our new compounds, AzoCholine, is an excellent photoswitchable agonist for neuronal α7 nAChRs, whereas BisQ was confirmed to be an agonist for the muscle-type nAChR. AzoCholine could be used to modulate cholinergic activity in a brain slice and in dorsal root ganglion neurons. In addition, we demonstrate light-dependent perturbation of behavior in the nematode, Caenorhabditis elegans.

Tissue slice cultures from humans or rodents: a new tool to evaluate biological effects of heavy ions.

The aim of this interdisciplinary project is to establish slice culture preparations from rodents and humans as a new model system for studying effects of X-rays and heavy ions within normal and tumor tissues. The advantage of such slice cultures relies on the conservation of an organotypic environment, the easy treatment and observation by live-imaging microscopy, and the independence from genetic immortalization strategies used to generate cell lines. Rat brains as well as human tumors were cut into 300-mum-thick sections and cultivated in an incubator in a humidified atmosphere at 37 degrees C. This is realized by a membrane-based culture system with a liquid-air interface. With this system, it is possible to keep rodent slices viable for several months. Human brain tumor slices remained vital for at least 21 days. Slices were irradiated with X-rays at the radiation facility of the University Hospital in Frankfurt/Main at doses up to 40 Gy. Heavy ion irradiations were performed at GSI (Darmstadt) with different ions, energies, and doses. The irradiated slices were analyzed by 3D-confocal microscopy following immunostaining for DNA damage, microglia, and proliferation markers. The phosphorylated histone gammaH2AX proved to be suitable for the detection of ion traversals in this system.